Imperial/Wet Lab/Lab Notebook/2007-08-14


14 August 2007

Preparation of Stock Solutions

  • 1x 1 litre of 2xYT medium in Duran bottle
  • 3x 1 litre of 2xYT medium in conical flask
    • Sent to autoclave
  • 1x 10 ml of pre-incubation solution for preparation of cell extract
  • 6x 20 ml of 1mM IPTG stock

Preparation of Cell Extract

  1. Picked a BL21(DE3) colony from Tet plate
  2. Innoculated in 100 ml of 2xYT + Tet medium
  3. Incubated overnight at 37 °C

Cloning of Biobricks

  1. Placed 5 ml of LB in a 15 ml tube
  2. Added appropriate antibiotics into the tube
  3. Picked a colony from the fresh overnight plate
  4. Innoculated the colony in the LB media
  5. Incubated for 6 hours during the day
    • 2x 6 Biobricks cloned

Cloning of Biobricks (for Midiprep)

  1. Placed 50 ml of LB in a 250 ml conical flask
  2. Innoculated 1 ml of culture into LB media in flask
  3. Incubated overnight at 37 °C

  • 2. BBa_I13422 [ptet-GFP]
  • 5. BBa_R0040 [ptet]
  • 7. BBa_F2620 [plux]
  • 8. BBa_F1610 [luxI]
  • 11. BBa_E7104 [pT7]
  • 13. BBa_B0015 [stop]
  • 14. BBa_R0051 [pcI]
  • 16. BBa_E0240 [GFP]
  • 17. BBa_I13507 [RFP]
  • 18. BBa_T9002 [plux-GFP]

Pilot Preparation of Vesicles

  • The suspension prepared the day before was used to produce vesicles enclosing Tris and NaCl buffer only.
  • There was a mistake in following the protocol: instead of preparing the interface with 2ml of the DOPC-mineral oil suspension and then adding 100μl of emulsion to it, the emulsion was prepared according to protocol, and then 2ml of emulsion was used to create the interface.
  • Results: A sample was briefly looked at under a light microscope, but no vesicles were observed.